The significance of cellular senescence for in vivo aging is not yet clear. The recent development of a greater understanding of the molecular mechanisms by which normal human cells achieve a terminally nonreplicating state in culture has provided new molecular tools required for an analysis of the significance of this state in tissues in vivo. In this Project, the cells of the adrenal cortex, which have previously been well characterized with respect to cellular senescence, will be used to investigate this relationship. 1. An in vivo model of adrenocortical regeneration and continuous passaging will be set up to establish whether normal adrenocortical cells from three species (human, bovine, and rat) can exhaust their proliferative potential in this in vivo model and to compare the proliferative potential of adrenocortical cells in vivo with their proliferative potential in culture. 2. Because telomere length appears to provide a good indicator of the proliferative age of cells in culture and in vivo, assays will be performed to establish whether changes in telomere length are similar in adrenal cells passaged maximally in vivo and in cells passaged maximally in culture. 3. Based on the observation that human, bovine, and rat adrenocortical cells do not normally express p21 senescent cell derived inhibitor (SDI1) in vivo, yet rapidly induce this gene when transferred to the culture environment, experiments will be performed to investigate the functional status of p21/Sdi1 in adrenocortical cells in culture and its effects on cell proliferation. 4. Experiments will be performed to investigate the cause of the high level of p21/Sdi1 expression in adrenocortical cells in culture. 5. Because p21Sdi1 is the only protein yet identified for which there is reason to believe that its expression in senescent cells is causative rather than associative with respect to the nondividing state, it is important to examine the expression and functional state of p21/Sdi1 in cells which have been maximally passaged in vivo and to compare it to that in cells that have reached the nondividing state by division in culture.